Abstract:

The invention relates to a method for the oxidative cleavage of vinyl
aromatics of the formula (1) characterized in that (a) compound(s) of the
formula (1) is/are oxidized to aldehydes and ketones of the formulas (2)
and (3), respectively, in the presence of molecular oxygen using at least
one enzyme selected from peroxidases and laccases as a catalyst,
according to the following general reaction scheme:
##STR00001##
wherein n is an integer of 0 to 5; the R1 are selected from saturated
or unsaturated hydrocarbon groups with 1 to 10 carbon atoms, wherein
carbon atoms are optionally substituted by heteroatoms and are optionally
further substituted, amino, C1-6 alkylamino and C1-6
dialkylamino groups, halogens, hydroxy and cyano, wherein two of the
substituents R1 may be linked to form a ring; R2 and R3
are each independently hydrogen or one of the options for R1,
wherein R2 and/or R3 may be linked with R1 to form a ring,
in which case R2 and R3 may each represent a chemical bond.

Claims:

1. A method for the oxidative cleavage of optionally substituted vinyl
aromatic compounds of the following formula (1) ##STR00014## comprising
oxidizing one or more compound(s) of formula (1) to aldehydes and ketones
of the formulas (2) and (3), respectively, ##STR00015## in the presence
of molecular oxygen, and as catalysts, at least one enzyme selected from
peroxidases and laccases, and at least one metalloprotein, according to
the following general reaction scheme: ##STR00016## wherein n is an
integer of 0 to 5, so that the aromatic ring in the compounds of formulas
(1) and (2) may be substituted at the ortho, meta and/or para position(s)
of the vinyl group with 0 to 5 substituents R1 which may be
identical or different and are selected from:a) saturated or unsaturated
hydrocarbon groups with 1 to 10 carbon atoms, wherein one or more carbon
atoms are optionally substituted by a heteroatom selected from oxygen,
nitrogen and sulfur, and which hydrocarbon groups are optionally further
substituted with one or more substituents selected from the group
consisting of C1-6 alkyl groups, C1-6 alkylene groups,
C1-6 alkoxy groups, amino, C1-6 alkylamino groups, C1-6
dialkylamino groups, halogens, hydroxy, oxo and cyano,b) amino, C1-6
alkylamino groups and C1-6 dialkylamino groups, andc) halogens,
hydroxy and cyano,wherein any two of the substituents R1 may be
linked to form an alicyclic or aromatic ring, andwherein the substituents
R2 and R3 are each independently hydrogen or one of the options
described in a), b) and c),wherein R2 and/or R3 in the formula
(1) compound(s) may be linked to a substituent R1 to form an
alicyclic ring, in which case R2 and R3 may each represent a
chemical bond between the carbon atom of the vinyl group to which they
are bound and the substituent R.sup.1.

2. The method of claim 1, wherein the at least one enzyme is selected from
fungal peroxidases and laccases, halogen peroxidases, lignin peroxidases,
horseradish peroxidase, and bovine milk peroxidase.

3. The method of claim 2, wherein the at least one enzyme is selected from
fungal peroxidases from Coprinus cinereus, from laccases from Coriolus
versicolor, Agaricus bisporus and Candida rugosa, laccase from Rhus
vernfera, chloroperoxidase from Caldariomyces fumago and bromoperoxidases
from Streptomyces aureofaciens or Corallina officinalis.

4. The method of claim 2, wherein the at least one enzyme is selected from
horseradish peroxidase, from peroxidases from Coprinus cinereus and
laccases from Coriolus versicolor and Agaricus bisporus.

5. The method of claim 1, wherein the oxidation is carried out in a
buffer.

6. The method of claim 5, wherein the buffer is selected from the group
consisting of Bis-Tris buffer, acetate buffer, formate buffer, and
phosphate buffer.

7. The method of claim 1, wherein the pH of the reaction is adjusted to 2
to 7.

8. The method of claim 1, wherein the oxidation is carried out at an
O2 overpressure.

9. The method of claim 8, wherein the oxidation is carried out at an
O2 overpressure of 1 to 6 bar.

10. The method of claim 1, wherein the oxidation is carried out under the
action of light.

11. The method of claim 1, wherein the oxidation is carried out in the
presence of an organic solvent or solvent mixture.

12. The method of claim 11, wherein the solvent or solvent mixture is used
at a content of 1 to 20% by volume of the reaction mixture.

13. The method of claim 11 or 12, wherein the organic solvent is selected
from the group consisting of C1-4 alkanols, dimethyl sulfoxide,
toluene, acetone, dioxane, tetrahydrofuran, dimethyl formamide, and
mixtures thereof.

14. The method of claim 1, wherein vanillin is produced as the aldehyde of
formula (2).

15. (canceled)

16. (canceled)

17. (canceled)

Description:

[0001]In continuation of this research, the present inventors have now
surprisingly found out that, under specific conditions, certain
peroxidases and laccases, and not only such of fungal origin, are able to
catalyze the oxidative cleavage by oxygen of special ethylenic double
bonds to aldehydes and ketones. This result was surprising since oxygen
is usually not a substrate (or in the case of laccases at least not a
preferred one) for such enzymes and the obtained oxidation products are
those usually obtained in ozonolytic reactions. For example, peroxidases
can, as the name implies, generally only process peroxide bonds, and
halogen peroxidases exclusively result in halogenated, e.g. chlorinated
or brominated, oxidation products.

DISCLOSURE OF THE INVENTION

[0002]The present invention provides a method for the oxidative cleavage
of ethylenic double bonds conjugated with aromatic rings, i.e. of
optionally substituted vinyl aromatics of the following formula (1), by
use of at least one metalloprotein as a catalyst, which is characterized
in that one or more compound(s) of formula (1) is/are oxidized to
aldehydes and ketones of the formulas (2) and (3), respectively, in the
presence of molecular oxygen using at least one enzyme selected from
peroxidases and laccases as catalyst, according to the following general
reaction scheme:

##STR00002##

wherein n is an integer of 0 to 5, so that the aromatic ring may be
substituted at the ortho, meta and/or para position(s) of the vinyl group
with 0 to 5 substituents

[0004]Due to current economic circumstances and an increasing
environmental awareness, the demand for mild and selective oxidation
methods as well as new ecological and economical chemical methods is
higher than ever before. The oxidative cleavage of alkenes into the
corresponding aldehydes and ketones is a synthetic method widely used in
organic chemistry (i) to introduce oxygen functionalities into molecules,
(ii) to split complex molecules into smaller units, and (iii) to remove
protective groups. Among the methods currently available for the chemical
oxidative cleavage of alkenes, reductive ozonolysis is regarded as the
"cleanest". In practice, however, this method has several disadvantages
such as the requirement of using special equipment (ozonator),
deep-temperature techniques (usually -78° C.), and the additional
need of stoichiometric amounts of reducing agents (e.g. dimethyl sulfide,
zinc, hydrogen, phosphines etc.) for the reductive treatment. In
addition, particular safety measures have to be taken in order to prevent
serious accidents, e.g. by explosions.

[0005]In other methods using metal oxides as oxidizing agents, (at least)
stoichiometric amounts of salts or peroxides are required. However, these
variations show moderate to low chemo-, regio- and stereoselectivities.
In many cases, overoxidation of the aldehydes obtained as intermediates
to the corresponding acids is a side reaction that is difficult to
prevent. For example, the use of OsO4 and NaIO4.sup.[1], of
OsO4 and Oxon® (2
KHSO5+KHSO4+K2SO4).sup.[2], of RuCl3 in
combination with NaIO4 or Oxon® .sup.[3], and of ruthenium
nanoparticles with NaIO4.sup.[4] have been described.

[0006]Consequently, an oxidation method for alkenes would be desirable,
which prevents the above disadvantages and, above all, uses a non-toxic,
easily available oxidizing agent such as oxygen.

[0007]However, the only known chemical-catalytic method that uses
molecular oxygen as an oxidizing agent requires a Co(II) compound as a
catalyst, is only moderately selective and is furthermore limited to
isoeugenol derivatives.sup.[5].

[0008]A possible alternative seems to be biocatalysis. However, enzymatic
alkene cleavages have only been described for a few, very specific
substrates, using a mixture of lipoxygenases and hydroperoxide
lyases.sup.[6].

[0009]In addition, the same cleavages have been described as undesired
side reactions, i.e. yielding oxidation products in analytical amounts,
in peroxidase-catalyzed processes.sup.[7]-[13]. Molecular oxygen, of
course, has not been used as oxidizing agent in any of these reactions.

[0010]Enzymatic alkene cleavage with oxygen by enzymatic catalysis has
also been attempted, but only with certain mono- and dioxygenases as
enzymes and with yields in analytical amounts.sup.[14]-[17]. In addition,
oxygenases have very high substrate specifities.sup.[18]-[29], so that
only a very limited selection of substrates can be used.

[0011]Against this background, the present inventors and their co-workers
had already found out in earlier research that certain aryl alkenes can
be oxidized to corresponding aldehydes and ketones, using molecular
oxygen as the oxidizing agent by adding cells or cell extracts of a
certain fungus, Trametes hirsute (white-rot fungus), which catalyze the
oxidation.sup.[30],[31]. Consequently, this is a biocatalyzed reaction,
probably by enzymatic catalysis. However, the enzyme(s) responsible
therefor could not be clarified.

[0012]In continuation of this research, the present inventors have now
surprisingly found out that, under specific conditions, certain
peroxidases and laccases, and not only such of fungal origin, are able to
catalyze the oxidative cleavage by oxygen of special ethylenic double
bonds to aldehydes and ketones. This result was surprising since oxygen
is usually not a substrate (or in the case of laccases at least not a
preferred one) for such enzymes and the obtained oxidation products are
those usually obtained in ozonolytic reactions. For example, peroxidases
can, as the name implies, generally only process peroxide bonds, and
halogen peroxidases exclusively result in halogenated, e.g. chlorinated
or brominated, oxidation products.

DISCLOSURE OF THE INVENTION

[0013]The present invention provides a method for the oxidative cleavage
of ethylenic double bonds conjugated with aromatic rings, i.e. of
optionally substituted vinyl aromatics of the following formula (1),
which is characterized in that one or more compound(s) of formula (1)
is/are oxidized to aldehydes and ketones of the formulas (2) and (3),
respectively, in the presence of molecular oxygen using at least one
enzyme selected from peroxidases and laccases as a catalyst, according to
the following general reaction scheme:

##STR00003##

wherein n is an integer of 0 to 5, so that the aromatic ring may be
substituted at the ortho, meta and/or para position(s) of the vinyl group
with 0 to 5 substituents R1 which may be identical or different and
are selected from: [0014]a) saturated or unsaturated hydrocarbon groups
having 1 to 10 carbon atoms, wherein one or more carbon atoms are
optionally substituted by a heteroatom selected from oxygen, nitrogen and
sulfur, and which are optionally further substituted with one or more
substituents selected from C1-6 alkyl groups, C1-6 alkylene
groups, C1-6 alkoxy groups, amino, C1-6 alkylamino and
C1-6 dialkylamino groups, halogens, hydroxy, oxo and cyano, [0015]b)
amino, C1-6 alkylamino and C1-6 dialkylamino groups, and
[0016]c) halogens, hydroxy and cyano,wherein any two of the substituents
R1 may be linked to form an alicyclic or aromatic ring, andwherein
the substituents R2 and R3 are each independently hydrogen or
one of the options described in a), b) and c),wherein R2 and/or
R3 may be linked to a substituent R1 to form an alicyclic ring,
in which case R2 and R3 may each represent a chemical bond
between the carbon atom of the vinyl group to which they are bound and
the substituent R1.

[0017]Thus, an oxidation method for the above compounds is provided, by
means of which the aim defined above can be achieved. This means that
aryl alkenes can be oxidized to the desired aldehydes and ketones such as
vanillin by use of oxygen, an omnipresent, harmless oxidizing agent, and
specific natural enzymes which are easily and economically available
through biological or biotechnological means. Thus, neither expensive or
toxic (heavy metal) catalysts nor complicated and expensive equipment
(ozonator, deep-temperature cooling systems) are required, and no waste
products with complicated disposal requirements are obtained.

[0019]Preferably, the method is carried out in a buffer in order to be
able to maintain stable reaction conditions, especially a stable pH,
during oxidation. Preferably, the reaction is conducted in Bis-Tris
buffer, acetate buffer, formate buffer or phosphate buffer. The pH value
of the reaction mixture is preferably adjusted to 2 to 7, more preferably
to 2 to 4, as the enzymes show their respective maximum activities in
these ranges.

[0020]In a further preferred embodiment, the inventive method is carried
out at an O2 overpressure to increase the yields. This, however, is
not simply the result of a shift of the reaction equilibrium, as can be
seen by the fact that some enzymes, having reached an activity maximum,
showed lower yields again at higher pressures. Preferably, oxidation is
carried out at an O2 overpressure of 1 to 6 bar, preferably 2 to 3
bar. Higher values do not or hardly result in additional improvements,
often even in lower conversions, and would considerably increase
equipment requirements. In the above pressure ranges, for example, a
conventional Parr apparatus can be used without difficulty.

[0021]In further preferred embodiments, the method is carried out under
the action of light, i.e. with irradiation, as this allows a multiple
increase of the yields, especially when laccases are used as enzymes.

[0022]In additional preferred embodiments, the method is carried out in
the presence of an organic solvent or solvent mixture, which is
preferably selected from C1-4 alkanols, dimethyl sulfoxide, toluene,
acetone, dioxane, tetrahydrofuran, dimethyl formamide and mixtures
thereof, and its content is preferably 1 to 20% by volume, more
preferably 5 to 15% by volume, of the reaction mixture.

[0023]In a further aspect, the invention thus concerns the use of fungal
peroxidases and laccases, fungal halogen peroxidases, bacterial halogen
peroxidases, lignin peroxidases, horseradish peroxidase or bovine milk
peroxidase for the catalysis of the oxidative cleavage of ethylenic
double bonds conjugated with the aromatic ring of optionally substituted
vinyl aromatics by molecular oxygen, wherein the same enzymes are
preferred as described for the method according to the first aspect of
the invention.

SHORT DESCRIPTION OF THE DRAWINGS

[0024]FIGS. 1 to 3 show the conversion changes in the inventive method
with the addition of various organic solvents.

[0025]The invention will now be described in more detail by means of
specific examples, which are provided for illustration purposes only and
not for limitation.

EXAMPLES

[0026]Once the reactivity of various enzymes as catalysts of the oxidation
of aryl alkenes by molecular oxygen had been determined in preliminary
experiments, the pH optimum of the individual enzymes for such reactions
was established in a model reaction using trans-anethole as the vinyl
aromatic of formula (1). For adjusting the pH to values from 2 to 7,
known buffer systems were used.

Examples 1 to 16

##STR00004##

[0028]The respective enzymes (3 mg each of the preparations, which were
all solid) were placed into the wells of a "Riplate LV" 5 ml Deep Well
Plate (HJ-Bioanalytik GmbH). Subsequently, 900 μl of the respective
buffers and 6 μl (0.04 mmol) of trans-anethole were added. The plates
were then placed into an O2 pressure reactor in an upright position.
The reactor was purged with pure molecular oxygen, and the pressure was
adjusted to 2 bar oxygen. After 24 h at 170 rpm and 25° C., the
reaction mixtures were transferred into 2 ml test tubes, and the wells
were washed with EtOAc (600 μl). These 600 μl were added to the
respective test tubes in order to also carry out a first extraction of
the aqueous reaction mixtures therewith. After a second extraction with
pure EtOAc (600 μl), the combined organic layers were dried over
Na2SO4 and analyzed for the conversion to p-anisaldehyde
(4-methoxy benzaldehyde) by GC.

[0031]This table clearly shows the surprising catalytic effect of the
peroxidases, halogen peroxidases and laccases tested in the above
oxidation reaction. Compared to halogen peroxidases and laccases,
peroxidases are more reactive, but the conversions of some other enzymes,
especially of Agaricus bisporus laccase, are absolutely sufficient for a
preparative implementation of the inventive method without the necessity
of carrying out one of the optimizations described further below.

[0032]Next, the effect of oxygen pressure on the performance of the
enzymes as biocatalysts was tested.

Examples 17-31

Oxidation of Trans-Anethole at Various Oxygen Pressures

[0033]Essentially, the reactions and GC measurements were carried out as
in Examples 1 to 16, with the exception that the pressure for each enzyme
tested was varied between 2 and 6 bar. Due to the extensive equipment
requirements, higher pressures were not examined. The results of the
tests are shown in the following Table 2.

[0034]It is noticeable that no general preference for higher or lower
pressures can be identified. The expectation that higher oxygen pressures
would shift the reaction equilibrium towards the products side was not
fulfilled. Rather, it seems that each enzyme does not only have an
optimum pH range, but also an optimum pressure range.

[0035]In further experiments, the various enzymes were tested with
different substrates, e.g. aryl alkenes of formula (1), under otherwise
substantially equal conditions in order to be able to deduce substrate
specifities. The only exception was that the reactions were carried out
at the respective enzymes' pH optima, as previously determined.

Examples 32 to 36

##STR00005##

[0037]The reactions, work-ups and GC measurements were conducted as
described for Examples 1 to 16 (2 bar oxygen) and using a buffer
corresponding to the respective pH optimum. The enzymes, buffers, pH
values used and the conversions of trans-anethole to p-anisaldehyde are
shown in the following Table 3.

[0038]Again, it was clearly shown that trans-anethole can be oxidized to
p-anisaldehyde by enzymatic catalysis with sometimes very good yields,
and that peroxidases are clearly superior to laccases, even though the
latter can also be used for preparative purposes.

Examples 37 to 40

##STR00006##

[0040]By analogy with Examples 32 to 36, 4-aminostyrene instead of
trans-anethole was oxidized to 4-aminobenzaldehyde, using different
enzymes and different buffers. In addition, during work-up, the pH of the
aqueous phase was adjusted to 10 in order to prevent salification of the
amino groups. The results of the experiments and the GC measurements are
shown in Table 4.

[0041]It has been shown that, under the experimental conditions, among the
four enzymes tested only laccase from Coriolus versicolor could catalyze
the oxidation of 4-aminostyrene with good conversion results. This fact
and the fact that the enzyme developed more than twice its efficiency
compared to the case of trans-anethole clearly prove that the enzymes
show substrate specifity.

##STR00007##

Example 41

[0042]By analogy with Example 32, 4-methoxystyrene instead of
trans-anethole was oxidized with the peroxidase from Coprinus cinereus,
batch 1, to p-anisaldehyde. The results of the two experiments are shown
in Table 5.

[0043]This example again gives evidence for the substrate specifity of the
enzymes. Obviously, the peroxidase tested is able to oxidize very well
the double bond of trans-anethole substituted on both sides, however,
hardly oxidizes that of methoxystyrene, being unsubstituted on one side.
Thus, the substituents of the vinyl group have a substantial effect on
the catalytic reaction.

Examples 42 and 43

##STR00008##

[0045]By analogy with Examples 32 and 33, 2-bromostyrene instead of
trans-anethole was oxidized with the peroxidase from Coprinus cinereus,
batch 1, and the horseradish peroxidase, batch 1, in this case to
2-bromobenzaldehyde. The results of the four experiments are shown in
Table 6.

[0046]This result again proves that substituents at the aromatic ring also
have a substantial effect on the catalytic reaction.

Example 44

##STR00009##

[0048]By analogy with Example 33, ω,ω-dimethylstyrene instead
of trans-anethole was oxidized with horseradish peroxidase, batch 1, in
this case to benzaldehyde. The results of the two experiments and those
of Example 43 are shown in Table 7 for comparative purposes.

[0049]Again, evidence for the substrate specifity was provided. The
presence of two methyl groups at the ω-carbon of styrene instead of
only one and the lack of substituents at the aromatic ring have a
substantial effect on the catalysis.

Examples 45 to 47

##STR00010##

[0051]By analogy with Examples 32, 33 and 36, indene instead of
trans-anethole was oxidized with the peroxidase from Coprinus cinereus,
batch 1, horseradish peroxidase, batch 1, and the laccase from Coriolus
versicolor, in this case to 2-(formylmethyl)benzaldehyde. The results of
the six experiments are shown in Table 8.

[0052]While the two peroxidases had clearly lower catalytic activity when
indene was used as a substrate, the (less active) laccase showed hardly
any difference compared to trans-anethole. However, it is proven that
ethylenic double bonds contained in cyclic structures may also be
oxidized according to the inventive method.

Comparative Examples 1 and 2

##STR00011##

[0054]By analogy with Examples 32 and 33, 5-o-tolyl-2-pentene instead of
trans-anethole was oxidized with the peroxidase from Coprinus cinereus,
batch 1, and horseradish peroxidase, batch 1, in this case to
3-o-tolylpropionaldehyde. The results of the two experiments are shown in
Table 9.

[0055]These results show that arylalkenes with non-conjugated double bonds
cannot be oxidized with the inventive method. The oxidation products are
only detectable in analytic amounts (≦1%).

Examples 48 to 51

Oxidation of Trans-Anethole Under Various Light Conditions

[0056]The reactions and GC measurements were conducted in duplicate for
each enzyme tested, essentially as described in Examples 32 to 36 in the
buffers indicated therein for the respective enzymes. However, in a first
experimental series, a lamp (PAR 38 EC Spot, Osram Concentra, 120 W, 230
V, 448) was arranged at a distance of 50 cm above the reactor in order to
illuminate the reaction mixtures during oxidation, while in a second
experimental series the reactor was covered with an aluminum foil for
darkening, which foil was perforated to allow oxygen exchange with the
environment. The results of the best four of all enzymes tested are shown
in the following Table 10.

[0057]The results show a clear increase of the catalytic activity of all
four enzymes under the action of light. This effect is particularly well
pronounced with the two laccases, since their effectiveness was increased
to the eight- or ninefold level. Thus, generally less active laccases can
also result in very good conversions for preparative purposes.

Examples 52 to 85

Oxidation of Trans-Anethole in the Presence of Various Organic Solvents

[0058]By analogy with the method described for Examples 1 to 16,
trans-anethole was oxidized by means of peroxidase from Coprinus
cinereus, batch 1, or horseradish peroxidase, batch 1, as a catalyst. The
conversions thus obtained to p-anisaldehyde of 44% and 58%, respectively,
served as blanks for subsequent repetitions of the methods, wherein,
however, in each case 17 μl of an organic solvent were added to the
900 μl of the aqueous trimethylammonium formate/formic acid buffer (20
mM).

[0059]The results are shown in the following Tables 11 and 12 and shown
graphically in FIGS. 1 and 2, wherein the horizontal lines show the
values of experiments without solvent ("blank").

[0060]All results prove that the presence of an organic solvent is
basically possible without completely inhibiting the oxidation reaction.
As may be seen from Table 11 and FIG. 1, peroxidase from Coprinus
cinereus is more sensitive to the addition of a solvent because the
conversions are all lower due to the solvents--with the exception of
DMSO.

[0061]In contrast, the addition of a solvent mostly leads to a conversion
increase when horseradish peroxidase is used as the catalyst. Only in the
cases of cyclohexanol and Tween 80, there is a decrease, and 2-propanol
provides the same result as the blank.

[0062]Without wishing to be bound by any special theory, it is assumed
that here the solvent serves as solubilizer for the compound of formula
(1) to be oxidized, even though only 1.8% by volume of a solvent were
added to the aqueous buffer. In order to investigate if higher amounts of
a solvent would also have a positive effect on the conversion using
horseradish peroxidase, a further test series was conducted with
increasing amounts of DMSO, the only solvent that had shown a positive
effect with peroxidase from Coprinus cinereus.

Examples 86 to 96

Oxidation of Trans-Anethole in the Presence of Increasing Amounts of DMSO

[0063]By analogy with the above Examples 69 to 85, trans-anethole was
oxidized with horseradish peroxidase as a catalyst, wherein the 900 μl
of aqueous buffer were replaced by increasing percentages of DMSO. In
Table 13, the conversions achieved at the respective contents of DMSO are
shown. In FIG. 3, the data are also shown graphically.

[0064]It may be seen that, at a content of 40% of DMSO in the medium,
horseradish peroxidase shows approximately the same activity as without
organic solvent. At higher concentrations the activity decreases quickly,
and from 60% of DMSO onward substantially no enzymatic effect is
detectable. With 20 to 30% of DMSO in the medium, the conversion was
increased by approximately 20%. The best results, i.e. a conversion
increase of approximately 40%, was achieved with 5 to 15% of the solvent.

[0065]To verify if this effect is also detectable in other reactions, the
following test series for producing vanillin
(4-hydroxy-3-methoxybenzaldehyde), one of the possible valuable reaction
products of the inventive method, was conducted.

Examples 97 to 104

##STR00012##

[0067]By analogy with Examples 52 to 85, isoeugenol
(2-methoxy-4-propene-1-ylphenol) instead of trans-anethole was oxidized
with peroxidase from Coprinus cinereus or horseradish peroxidase as
catalyst. The aqueous buffer was replaced by 10 to 20% by volume of DMSO.
The results are shown in the following Table 14.

[0068]It has been shown that, with Coprinus cinereus peroxidase, DMSO
resulted in a conversion increase of approximately 15-20% in all
concentrations tested, while horseradish peroxidase hardly benefited from
the presence of DMSO in this reaction (i.e. max. 5%).

Examples 105 to 114

##STR00013##

[0070]By analogy with Examples 97 to 104, coniferyl alcohol
(4-hydroxy-3-methoxy cinnamyl alcohol) instead of isoeugenol was oxidized
using the two enzymes, which provides an alternative synthetic route for
obtaining vanillin by use of the inventive method and additionally
provides glycolaldehyde (hydroxyacetaldehyde) as a side product, which is
a useful reagent in protein chemistry. The aqueous buffer was again
replaced, in this case by 5 to 20% DMSO. Table 15 shows the results
obtained.

[0071]This clearly shows several facts. On the one hand, coniferyl alcohol
was quantitatively oxidized to vanillin by use of either of the two
enzymes as a catalyst, as long as no organic solvent was contained. On
the other hand, while horseradish peroxidase still tolerated 5% DMSO well
(6% decrease) and only showed clear activity losses afterwards (40 to
70%), Coprinus cinereus peroxidase decreased to 50% of its activity at
only 5% DMSO. At 15% DMSO, approximately 90% of its activity were lost.
These results are again gives evidence for the high specifity of the
enzymatic catalysts in the method of the present invention.

[0072]Thus, the effectiveness of the enzymes in the inventive method has
been clearly demonstrated. Based on the above teachings, the average
artisan can easily determine the optimal conditions regarding pH,
pressure, action of light, and organic solvent for individual, specific
enzymes in specific oxidation reactions by means of optimization series.
The invention thus constitutes an important contribution to the field of
biocatalysis for the production of organic compounds.